The location of origins of DNA replication within the Saccharomyces cerevisiae genome is primarily determined by the origin recognition complex (ORC) interacting with specific DNA sequences. The analogous situation in vertebrate cells is far less clear, although ORC subunits have been identified in several vertebrate organisms including Xenopus laevis. Monoclonal antibodies were raised against Xenopus Orc1p and used for single-step immunoaffinity purification of the entire ORC from an egg extract. Six polypeptides ( approximately 110, 68, 64, 48, 43, and 27 kDa) copurified with Xenopus Orc1p. Protein sequencing also showed the 64-kDa protein to be the previously identified Xenopus Orc2p. Microsequencing of the 43- and 48-kDa proteins that copurified with Orc1p and Orc2p led to their identification as the Orc4p and Orc5p subunits, respectively. Peptide sequences from the 43-kDa protein also allowed the isolation of cDNAs encoding the Xenopus, mouse, and human ORC4 subunits. Human ORC5 was also cloned; its sequence displayed extensive homology to both Drosophila and yeast ORC5. Surprisingly, comparison of the amino acid sequences of Orc1p, Orc4p, and Orc5p suggests that they are structurally related to each other and to the replication initiation protein, Cdc6p. Finally, we present the sequence of the putative Xenopus and human Orc3p.

• PMID: 9829972
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Tugal T, Zou-Yang XH, Gavin K, Pappin D, Canas B, Kobayashi R, Hunt T, Stillman B

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